Cell surface-bound La protein regulates the cell fusion stage of osteoclastogenesis

Multinucleated osteoclasts, essential for skeletal remodeling in health and disease, are formed by the fusion of osteoclast precursors, where each fusion event raises their bone-resorbing activity. Here we show that the nuclear RNA chaperone, La protein has an additional function as an osteoclast fusion regulator. Monocyte-to-osteoclast differentiation starts with a drastic decrease in La levels. As fusion begins, La reappears as a low molecular weight species at the osteoclast surface, where it promotes fusion. La’s role in promoting osteoclast fusion is independent of canonical La-RNA interactions and involves direct interactions between La and Annexin A5, which anchors La to transiently exposed phosphatidylserine at the surface of fusing osteoclasts. Disappearance of cell-surface La, and the return of full length La to the nuclei of mature, multinucleated osteoclasts, acts as an off switch of their fusion activity. Targeting surface La in a novel explant model of fibrous dysplasia inhibits excessive osteoclast formation characteristic of this disease, highlighting La’s potential as a therapeutic target.

The manuscript by Whitlock and colleagues describes the discovery of a significant novel function of the mammalian La protein. The mammalian full length La protein has previously been well characterized as an RNA binding protein with roles in the processing and metabolism of a variety of RNAs in the nucleus and cytoplasm. Through a series of experiments, the authors elucidated that a protease-cleaved form of La has an independent function at a different time and in a different location (on the cell surface) where it acts as an important regulator of osteoclast membrane fusion. They also discuss that the shorter form of La could potentially be a significant new therapeutic target for diseases that involve bone remodeling, for example, osteoporosis. The protein is also an interesting example of a protein with two very different functions in different cellular localizations, which would be of interest to many labs interested in understanding and predicting protein functions and regulation in general. Although I do not personally have experience in some of the methods that were used, in my opinion the data analysis, interpretation, and conclusions appear to be accurate and described with enough detail to be reproduced. My suggestions are mainly grammatical: p. 3 -traffics -> trafficks as verb p. 10 -2nd paragraph -osteoclasts misspelled p. 12 first line, add word "but" after comma p.12 3rd paragraph -concentrations of RANKL p. 15 middle of 2nd paragraph -Anx A1, which …. Anx A4, which …. p. 15 protein complexes further support the p. 17 2nd paragraph -low in osteoclasts precursors, precursors maintain very low levels = repetitive p. 19 2nd line -targets instead of approaches Various places throughout -commas needed to split sentence in 2 after "and", other places "the" or "a" sometimes need to be added Methods -E in E. coli should be capitalized, Thermo should probably be ThermoFisher, p. 5 in methods -sterile is misspelled as steril Antibodies section -please check positions of parentheses, check for "a" where it should be the letter alpha in a few places (p. 6) p. 7 stoichiometrically is misspelled p. 10 Alexa Fluor is misspelled Reviewer #3 (Remarks to the Author): The authors report that the spatial-temporal regulation of La protein contributes to monocyte-toosteoclast differentiation. While La protein is typically thought to play roles in RNA metabolism, the manuscript describes a new role of cell-surface La protein in osteoclast formation. Upon phosphorylation at S366 and caspase cleavage, a low molecular weight species of La is present at the cell surface. Proteomics identified that the levels of this low-molecular weight species of La correlates with multinucleated osteoclasts. This novel La function appears to be independent of the canonical N-terminal binding domains of La proteins. Through biochemical and cellular approaches, the authors show that fusion can be regulated by the levels of La. Moreover, the authors show an 4C A7A; 9;348 ;5 ;>?4;280>? 5;=90?7;: 7: 571=;@> 3D><80>70 3=7A4: 1D 9@?0?7;:> 7: (E>& /7?6 ?67> model, the manuscript shows that the application of anti-La antibodies can reduce osteoclast fusion, suggesting a possible use of La protein as a potential therapeutic target. The findings reported by the manuscript authors are noteworthy and of interest to the field. More information or clarification should be added to ensure the data is presented clearly. Major Comments: 1)"Since La, on its own, initiates neither hemifusion nor fusion between bound membranes, it is unlikely that La works as a "blue collar worker" directly catalyzing and/or driving membrane fusion. More likely La, as a "white collar worker", recruits or stimulates other components of the osteoclast fusion complex." •The authors are inappropriately referring to proteins in a manner that perpetuates inequities in our field. Referring to proteins as "blue collar" and "white collar" is unnecessary and vague. Rather they should be referred to as direct or indirect actions.
2)The use of technical replicates and biological replicates is unclear in the manuscript, along with the use of statistics • Figure 1b. SEM is represented as bars in figure 1b and can be misleading. It is better to represent data as a range on the graph. There are no asterisks visible in the figure, but a sentence regarding the symbol and the statistical test is present in the figure legend.
• Figure S2, Figure S5d. Bars (n=2) are represented on the graph without a description of what they refer to. Readers may assume these are SEM or SD values. The authors should clearly indicate that these are ranges. •In Figure 4, Figure 3g: statistics are reported for two replicates when one cannot determine a normal distribution from two measurements. Minor Comments: 1)The manuscript stressed the N-terminal RNA binding domains of La, naming the triple mutant "F- +'#& .; 2;:57=9 ?60? ?64 5@:2?7;: ;5 *0 7: ?67> 2;:?4C? 7> -+'%7:34<4:34:?$ ?64 ), <@88 3;B:> with La should be performed with RNAses. The authors should discuss the C-terminal RRM included in their constructs and how it may play novel La-RNA binding roles in their model. It is difficult to determine that the role of La in osteogenesis is independent of RNA as the protein may have different RNA binding residues presented on the cell surface. 2)Either figure S1d incorrectly refers back to Figure 1b, or the connection between the data is unclear. 3) Figure S1 panel lettering is off. 4) Figure 7c is not called out in the text. 5)The use of a period after Doxy. makes reading the manuscript difficult. I suggest using Doxy as a substitute. 6)It is unclear how many technical and biological replicates were used in the tandem mass-spec data. The table representing six samples (S1d should be b, see point 3) and the description in the figure legend is confusing. It appears the samples were taken from two biological replicates with three technical replicates each. The language should be clarified. Additional information should be added to the methods as to how the mass-spec data was filtered. 7)"La's tight regulation during osteoclastogenesis must be carried out at the protein level, as, despite the scarcity of La protein, M-CSF derived precursors contain even more La transcript (gene SSB) than after RANKL application (Fig. S1e)." •The authors have shown that osteoclastogenesis relies on the appropriate post-translational species and trafficking of La-protein. However, they cannot conclude that other forms of posttranscriptional regulation contribute more to these events without the appropriate controls. I suggest softening the language for this sentence.
Reviewer #4 (Remarks to the Author): The manuscript by Whitlock et al describes the involvement of La protein (SSB) in osteoclast fusion. Osteoclasts are essential for skeletal remodelling due to bone-resorbing properties, and derive from monocytes upon stimulation with cytokines including M-CSF and RANKL. By monitoring changes in protein levels during monocyte differentiation in vitro, the authors discover an involvement of the RNA binding protein La, which they aim to characterise in the rest of the study. Although understanding the molecular mechanism of regulated osteoclast fusion is a very important outstanding question in the field, the current study lacks clarity, fails to present a convincing mechanism of La-protein action and moreover suffers in part from poor experimental design and lack of experimental controls. Thus, the present study and conclusions drawn seem rather premature and add confusion instead of offering new insights into the biology of osteoclast fusion. Moreover, the manuscript has not been assembled with sufficient care (e.g. an antibody annotation conflict Fig 1D, and incorrect in-text references to Fig3).
Major concerns 1) A more unbiased quantitative approach seems warranted to monitor proteomic changes during osteoclast differentiation/fusion. 2) Given the central role of different molecular species of La protein, which the authors claim to detect by using "specific" antibodies, the antibodies in question must be validated more carefully. The fact that La protein exhibits a different MW on blots presented in figures 1 and 2 (inconsistent use of MW standards) is very confusing. Furthermore, it is unclear if the blot in 1d is for La protein or rather RANK (Abcam 13918) as stated in the figure legend. Figure 1d and 1e are supposed to be blotted for La with the same antibody, those should be run on the same gel/blot in order to give a clear idea of steady-state FL and cleaved La species during the course of osteoclast formation. In order to specifically detect FL La, the authors use a phospho-specific antibody (abcam 61800), but fail to provide evidence (on a WB) that this indeed detects specifically the full-length protein. Thus all claims made in this regard seem highly questionable. More surprisingly the authors claim to detect (the cleaved form of) La at the cell surface. However, given the absence of a previously reported or experimentally addressed mechanism of La-protein secretion as well as the strikingly different cell surface staining pattern in human and mouse cells (Fig 3d-c) better specificity controls are required (e.g. does RNAi abolish/reduce staining). Also the Antibody PA5-29763 (which is referred to as a-LMW La) is actually raised against AA 2-242 of human SSB, and so should detect both the long and short form of the protein. 3) In order to evaluate the more canonical role of RNA-binding protein La in post-transcriptional regulation of mRNAs, the authors evaluate mRNA levels of know osteoclast fusion factors. However, the literature and even the publication cited by the authors (Sommer et al 2011) mainly describes a role of La in mRNA translation. Thus, the authors must look at protein and not mRNA levels to reach a valid conclusion. 4) In figure2, the fusion promoting effect of ectopic La expression should best be evaluated in the RNAi background. This type of rescue experiment would additionally validate RNAi specificity. Additionally, the authors have to provide WB analysis of different constructs. mRNA/RT-qPCR is insufficient and likely flawed by the large excess of input/transfected DNA. 5) As mentioned above the cell surface staining with anti-La presented in Figure 3 requires further validation. Furthermore, the use of abs to inhibit fusion has to be controlled better. It is not clear that the observed effect is not due to high (background) binding by anti-La, where control abs do not bind, or bind to a much lesser extent. 6) It remains unclear if the fusion stimulating effect of recombinant La (Fig.4) is exerted on the cell surface and not inside the cell. Certainly, the prolonged (overnight) incubation time with recombinant protein would allow for cellular uptake and a cytosolic effect. Thus, further control experiments are warranted. 7) The authors have addressed whether La acts itself as a fusogen. This is described only in the text but the underlying data is not shown/has been omitted? 8) In order to evaluate a role for La as a fusion regulator the authors describe its interaction with Annexin A5 (a protein previously reported by the same lab to be involved in fusion). However, data presented in Fig S5 are not convincing as lysate controls are missing or not run properly alongside the immuno-precipitated samples. The data representing liposome binding experiments (FigS5 D) are confusing in terms of what has been tested but essential negative controls seem to be missing. Also, the primary data (protein gels should be shown). More importantly however, the authors fail to provide any evidence that would help to answer the question of how the postulated interaction with Annexin A5 would stimulate osteoclast fusion. 9) Given insufficiently controlled RNAi and anti-La surface binding/inhibition of fusion experiments, effects observed by the authors on bone resorption ( Fig 5) in a osteoblast/osteoclast co-culture setting (Fig 6) and in a mouse model exhibiting an excess of osteoclast fusion (Fig 7), do not really add to the story, as unfortunately they cannot be interpreted with sufficient confidence. binds to Anx A5 is now supported by following lines of evidence: (1) La-containing protein complexes isolated by immunoprecipitation from osteoclast lysates contain Anx A5; (2) Anx A5-containing protein complexes contain La; (3) La binding to liposomes depends on Anx A5, Ca 2+ and PS; (4) streptavidin pulldown of 6xHis-La from mixtures of recombinant Biotin-AnxA5 and 6xHis-La; (5, 6) loss of cell surface La observed after lowering the cell surface Anx A5 by either Ca 2+ removal or suppressing PS cell surface exposure. Our new findings (items 4, 5 and 6 above) provide additional evidence for La-Anx A5 binding, explain how La, a soluble protein, associates with cell membrane, and connect La function in osteoclastogenesis with non-apoptotic phosphatidylserine exposure signaling pathways that regulate osteoclast fusion.
We agree that the mechanism by which La promotes fusion remains to be understood. It could take many years and work of many labs to resolve this mechanism. As an analogy ,  remain to be clarified, we do consider our discovery of the regulatory role of La in the cell fusion stage of osteoclastogenesis to be a major advance and finding that osteoclast fusion is regulated by cell-surface complexes of La and Anx A5 bound to phosphatidylserine to be an important mechanistic insight. Moreover, this entirely novel function for a ubiquitous, nuclear RNA chaperone was completely unexpected, and demonstrates that evolution has repurposed La for alternative, noncanonical functions outside the cell. Furthermore, our findings identify easily accessible cellsurface La as an intriguing target for treating bone loss diseases. 3. In Fig.S4, the authors should check the effect of RNAi suppression of La on the expression levels of other key fusogenic factors such as DC-STAMP, OC-STAM, ATP6v0d2 and Sbno2.

The conclusion that RNAi suppression of La inhibits fusion by lowering cell surface La rather than by a loss of other factors is now supported by finding that fusion inhibition by La targeting RNAi is partially rescued by application of recombinant La (Fig. S4e).
Ea TWW\g\ba+ \a eXfcbafX gb g[X NXi\XjXetf VbaVXea TaW Tg g[X suggestion of Reviewer #4 we have added Figure  NFATc1 expression does not change as a result of La suppression. We did attempt to assess OC-STAMP levels using a commercially available, previously published antibody, but unfortunately this reagent did not produce any signal in control or La suppressed conditions, leaving us unable to comment on OC-STAMP. We do not consider adding more proteins to the list of tested as critical for the paper because our data show rapid inhibition and promotion of synchronized fusion by antibodies and recombinant proteins added to already differentiated osteoclast precursors. These findings show that La functions downstream of pre-fusion differentiation steps. 4. Given that La is ubiquitously expressed in various cell types, it is interesting to know whether La functions as a fusogenic factor only in osteoclasts. The authors should check the effect of La deletion on myocyte fusion and discuss molecular mechanisms underlying the cell type-dependent functions of La protein.
In revision we have added new data suggesting that mechanisms underlying cell surface La promotion of osteoclast fusion are not shared in myoblast fusion (Fig. S7c,d). Specifically, we found that application of z-La antibodies 'z-La mAb) and recombinant La (La 1-375 La*) (the reagents that inhibited and promoted osteoclast fusion, respectively) to differentiating C2C12 myoblasts have no effect on myoblast fusion.

REVIEWER 2.
We greatly appreciate the suggestions that helped us to strengthen the manusrcipt.
My suggestions are mainly grammatical: p. 3 traffics -> trafficks as verb p. 10 2nd paragraph osteoclasts misspelled p. 12 first line, add word but after comma p.12 3rd paragraph concentrations of RANKL p. 15 middle of 2nd paragraph Anx A1, which . Anx A4, which . p. 15 protein complexes further support the p. 17 2nd paragraph low in osteoclasts precursors, precursors maintain very low levels = repetitive p. 19 2nd line -targets instead of approaches Various places throughout commas needed to split sentence in 2 after and , other places the or a sometimes need to be added Methods E in E. coli should be capitalized, Thermo should probably be ThermoFisher, p. 5 in methods sterile is misspelled as steril Antibodies section please check positions of parentheses, check for a where it should be the letter alpha in a few places (p. 6) p. 7 stoichiometrically is misspelled p. 10 Alexa Fluor is misspelled

REVIEWER #3
We greatly appreciate the critiques and suggestions that helped us to strengthen the manusrcipt.
RNA metabolism, the manuscript describes a new role of cell-surface La protein in osteoclast formation. Upon phosphorylation at S366 and caspase cleavage, a low molecular weight species of La is present at the cell surface. Proteomics identified that the levels of this low-molecular weight species of La correlates with multinucleated osteoclasts. This novel La function appears to be independent of the canonical N-terminal binding domains of La proteins. Through biochemical and cellular approaches, the authors show that fusion can be regulated by the levels of La. Moreover, the authors show an ex vivo model of osteoclast formation in fibrous dysplasia driven by mutations in G s. With this model, the manuscript shows that the application of anti-La antibodies can reduce osteoclast fusion, suggesting a possible use of La protein as a potential therapeutic target. The findings reported by the manuscript authors are noteworthy and of interest to the field. More information or clarification should be added to ensure the data is presented clearly.
Major Comments: 1) Since La, on its own, initiates neither hemifusion nor fusion between bound membranes, it is unlikely that La works as a blue collar worker directly catalyzing and/or driving membrane fusion. More likely La, as a white collar worker , recruits or stimulates other components of the osteoclast fusion complex. The authors are inappropriately referring to proteins in a manner that perpetuates inequities in our field. Referring to proteins as blue collar and white collar is unnecessary and vague. Rather they should be referred to as direct or indirect actions.
Thanks, Agreed, we have changed the language as suggested.
2)The use of technical replicates and biological replicates is unclear in the manuscript, along with the use of statistics Figure 1b. SEM is represented as bars in figure 1b and can be misleading. It is better to represent data as a range on the graph. There are no asterisks visible in the figure, but a sentence regarding the symbol and the statistical test is present in the figure legend.
Agreed. SX haWXefgTaW g[X NXi\XjXetf concern and have replaced the previous graph with one now representing each replicate at each timepoint with dots. We hope that this will more clearly relates the temporal relationship of RANKL addition and osteoclast formation that we observe, as well as fully illustrates the variability observed between cells donated by different individuals. We have corrected the sentence concerning asterics and statistical tests, please forgive our oversight. Minor Comments: 1)The manuscript stressed the N-terminal RNA binding domains of La, naming the triple mutant ( RNA). To confirm that the function of La in this context is RNA-independent, the IP pull downs with La should be performed with RNAses. The authors should discuss the Cterminal RRM included in their constructs and how it may play novel La-RNA binding roles in their model. It is difficult to determine that the role of La in osteogenesis is independent of RNA as the protein may have different RNA binding residues presented on the cell surface.
P[Ta^f" SX TZeXX g[Tg aT`\aZ g[X ge\c_X`hgTag {NJ= j\g[bhg W\eect analysis of RNA binding has been inappropriate. In the revised MS, we have renamed this mutant into La*. We agree that we cannot exclude a novel role of RRM2 bound RNA in the structure and function of cell surface LA and we added the corresponding comment to our discussion. rO\aVX eXVXag fghW\Xf YbhaW NJ=f ceXfXag ba g[X fheYTVX bY _\i\aZ VX__f g[Tg TeX \aib_iXW \a monocyte interactions 62, 63, \g eX`T\af cbff\U_X g[Tg NJ= \f \aib_iXW \a fheYTVX HTtf eb_X \a regulating osteoclast fusion. However, even if the function of La in osteoclast formation depends on La-RNA interactions at the cell surface through some yet unknown mechanism, possibly involving RRM2 30 , this novel function fundamentally differs from the classical functions of La dependent on its La domain-and RRM1-mediated RNA binding in the ahV_Xhf TaW Vlgbc_Tf`-r 2)Either figure S1d incorrectly refers back to Figure 1b, or the connection between the data is unclear. We thank the Reviewer for drawing our attention to an incorrect reference in our supplementary figure. This has been corrected so that the supplementary figure now refers to the silver gel with the band of interest denoted by an arrow in Figure 1c. 3) Figure S1 panel lettering is off. Figure S1 has been updated. Figure 7c is not called out in the text. SX [TiX iXe\Y\XW g[Tg g[\f Y\ZheX \f eXYXeXaVXW \a g[X Yb__bj\aZ fXagXaVX9 rDoxy.-induced osteoclastogenesis was accompanied by a ~17-fold increase in mRANKL produced by the explants (Fig. 7c).

5)
The use of a period after Doxy. makes reading the manuscript difficult. I suggest using Doxy as a substitute. Thanks, Doxy. has been replaced with Doxy throughout the MS. 6)It is unclear how many technical and biological replicates were used in the tandem mass-spec data. The table representing six samples (S1d should be b, see point 3) and the description in the figure legend is confusing. It appears the samples were taken from two biological replicates with three technical replicates each. The language should be clarified. Additional information should be added to the methods as to how the mass-spec data was filtered.
We thank the Reviewer for their critique. Please see where we have added to the legend r@TgT eXceXfXag gjb U\b_bZ\VT_ eXc_\VTgXf '0&1( TaW g[eXX gXV[a\VT_ eXc_\VTgXf 'T-V(-s Moreover, please see where we have added technical information on how we performed the mass spectrometry analysis. r>TaWf bY \agXeXfg jXeX Vhg Yeb`f\_iXe fgT\aXW ZX_f+ W\fgT\aXW+ subjected to in-gel tryptic digest and evaluated by liquid chromatography coupled with tandem mass spectrometry (Proteomics Core, NHLBI). Mass spectrometry analysis was performed using a Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) coupled to a Dionex UltiMate 3000-nLC (Thermo Scientific) liquid chromatography system. The data were searched using Sequest within Proteome Discoverer software v1.4 (Thermo Fisher Scientific) against the the UniProt human database. The results were filtered with a 1% false discovery rate at the level of proteins and peptides. Raw files were analyzed using the Mascot search engine (v2. 3 La-protein. However, they cannot conclude that other forms of posttranscriptional regulation contribute more to these events without the appropriate controls. I suggest softening the language for this sentence.

Thanks.
We have edited this sentence to: rSX fhZZXfg g[Tg g\Z[g eXZh_Tg\ba bY HT Whe\aZ osteoclastogenesis is carried out at the protein level, as, despite the scarcity of La protein, M-CSF derived precursors contain even more La transcript (gene SSB) than after RANKL Tcc_\VTg\ba 'B\Z-O0X(-s REVIEWER 4. We greatly appreciate the critiques and suggestions that helped us to strengthen the manusrcipt. The manuscript by Whitlock et al describes the involvement of La protein (SSB) in osteoclast fusion. Osteoclasts are essential for skeletal remodelling due to bone-resorbing properties, and derive from monocytes upon stimulation with cytokines including M-CSF and RANKL. By monitoring changes in protein levels during monocyte differentiation in vitro, the authors discover an involvement of the RNA binding protein La, which they aim to characterise in the rest of the study. Although understanding the molecular mechanism of regulated osteoclast fusion is a very important outstanding question in the field, the current study lacks clarity, fails to present a convincing mechanism of La-protein action and moreover suffers in part from poor experimental design and lack of experimental controls. Thus, the present study and conclusions drawn seem rather premature and add confusion instead of offering new insights into the biology of osteoclast fusion. Moreover, the manuscript has not been assembled with sufficient care (e.g. an antibody annotation conflict Fig 1D, and incorrect in-text references to Fig3). Fig. 1D (it should be anti-La Abcam 75927) and incorrect reference to the figure 3, both are corrected in the revised MS.

Sorry for the wrong annotation of the Ab in the
We agree that the molecular mechanism by which La promotes osteoclast fusion remains unclear. However, as noted in our reply to the 2 nd comment of Rev. 1, we do consider the discovery of the regulatory role of La in osteoclastogenesis to be a major advance in the field and finding that osteoclast fusion is regulated by cell-surface complexes of La and Anx A5 connecting La function with phosphatidylserine signaling in fusion-committed cells to be an important and unexpected mechanistic insight. Furthermore, our findings identify easily accessible cell-surface La as an intriguing target for treating bone loss diseases. Complete dissection of the mechanisms of osteoclast fusion will likely take many follow up studies. Above (reply to the 2 nd comment of Rev. 1) we mention an example of myomaker, for which 9 years after the discovery of myoblast fusion dependence on myomaker (Millay et al., Nature, 2013), we still do not know the underlying mechanisms.
Major concerns 1) A more unbiased quantitative approach seems warranted to monitor proteomic changes during osteoclast differentiation/fusion.

While we agree that unbiased quantitative study of proteomic changes during osteoclast differentiation/fusion will be useful for the field, this is not the goal of this work. We have identified La as a player at the early stage of our analysis and concentrated all our efforts and resources on it.
2) Given the central role of different molecular species of La protein, which the authors claim to detect by using "specific" antibodies, the antibodies in question must be validated more carefully. The fact that La protein exhibits a different MW on blots presented in figures 1 and 2 (inconsistent use of MW standards) is very confusing. Furthermore, it is unclear if the blot in 1d is for La protein or rather RANK (Abcam 13918) as stated in the figure legend. Figure 1d and 1e are supposed to be blotted for La with the same antibody, those should be run on the same gel/blot in order to give a clear idea of steady-state FL and cleaved La species during the course of osteoclast formation. In order to specifically detect FL La, the authors use a phospho-specific antibody (abcam 61800), but fail to provide evidence (on a WB) that this indeed detects specifically the full-length protein. Thus all claims made in this regard seem highly questionable. More surprisingly the authors claim to detect (the cleaved form of) La at the cell surface. However, given the absence of a previously reported or experimentally addressed mechanism of La-protein secretion as well as the strikingly different cell surface staining pattern in human and mouse cells (Fig 3d-c) better specificity controls are required (e.g. does RNAi abolish/reduce staining).
Also the Antibody PA5-29763 (which is referred to as a-LMW La) is actually raised against AA 2-242 of human SSB, and so should detect both the long and short form of the protein.
We are especially grateful to this Reviewer for drawing our attention to our confusing discussion of the anti-La antibodies used in our work. We are sorry that the validation of the antibodies (Abs) used in our studies has not been clearly explained.
Validation of the antibodies. The key conclusions of the paper are supported by Western blot analysis, fluorescence microscopy, fusion and bone resorption assays using monoclonal `he\aX z -HT Tag\UbWl 'z -La mAb; Abcam, 75927). The specificity of this Ab (z-La mAb; (Fig. S1 and Fig. 1). (3) La immunoprecipitated with this antibody is recognized by another commercially available anti-La antibody raised against a different region of La (Fig. 5a). '3( j[\_X z-La mAb shows us cytosolic and cell surface staining in osteoclasts at the time of fusion, the same antibody gives expected nuclear staining in HeLa cells (Fig. S1f). We have included a specific discussion of the validation of this antibody into the revised MS. (Fig. 1), appearance of La at the surface of fusing osteoclasts ( (Fig. 3 (Fig. 6).

In a few experiments we have used two other anti-La antibodies: rabbit polyclonal Abcam 61800 antibody that specifically recognizes phosphorylated human SSB/La (phosphoSer366) and rabbit polyclonal ThermoFisher antibody PA5-29763 raised against AA 2-242 of human SSB/La. The comments of this Reviewer have convinced us that our discussion of these Abs and the ways we named them (FL La Ab and a-LMW La Ab) were confusing and misleading. Our findings (mostly correlations between appearance and disappearance of different species of La in cell lysates and nuclear vs cytosolic localization) and the literature (the dependence of La cleavage on its dephosphorylation) supported but did not prove that Abcam 61800 preferentially recognizes FL-La and PA5-29763 preferentially recognizes LMW-La. As noted by the Reviewer PA5-29763 should detect both the long and short form of the protein. Our finding that PA5-29763 preferentially stains cytosolic and dephosphorylated La likely reflects modification of the cleaved protein. This empirically observed distinction between preferred specificities of Abcam 61800 and PA5-29763 to FL-La and LMW-La, respectively, is not important for our work. Changes in the levels and species of La and La localization from cytoplasm/cell surface to nucleus during osteoclast differentiation have been documented with z-La mAb; Abcam, 75927 that recognizes all species of La studied. In revision, we have renamed Abcam 61800 and PA5-29763 to avoid placing the suggested interpretation into the name of the reagents. Rabbit Ab Abcam 61800 specific for La phosphorylated at Ser366 is now referred to as -p366 La rAb (instead of -FL La in the original version). Rabbit Ab ThermoFisher PA5-29763 is now referred to as -La rAb (instead of -LMW La). We have also correspondingly modified the discussion of the experiments with these Abs.
In response to the differences in molecular weight standards, we apologize. Over the last three years during this work a variety of reagents became difficult to obtain due to global supply chain issues. We initially used a molecular weight standard from Bio Rad and substituted a standard from Biodynamics that our supply center was able to obtain so that we could move forward with our work. While the standards are roughly equivalent, each differs by 5-8 KDa from Biorad.
In response to the differences in molecular weight between Figure 1 and Figure 2, all Westerns from Figure 1 Figure 2 represents samples from an immortalized murine cell line. We find that human vs mouse La does exhibit a slight molecular weight difference. This empirical observation may be explained by mouse La containing an additional 7 amino acids or some difference in posttranslational modifications in human vs mouse osteoclasts. While human and mouse La share substantial sequence similarity, there are substantial differences in the amino acid sequences of the two homologues. In particular, the primary amino acid sequence of the cterminal half of La is rather divergent when comparing the two. 3) In order to evaluate the more canonical role of RNA-binding protein La in post-transcriptional regulation of mRNAs, the authors evaluate mRNA levels of know osteoclast fusion factors. However, the literature and even the publication cited by the authors (Sommer et al 2011) mainly describes a role of La in mRNA translation. Thus, the authors must look at protein and not mRNA levels to reach a valid conclusion.

4)
In figure2, the fusion promoting effect of ectopic La expression should best be evaluated in the RNAi background. This type of rescue experiment would additionally validate RNAi specificity. Additionally, the authors have to provide WB analysis of different constructs. mRNA/RT-qPCR is insufficient and likely flawed by the large excess of input/transfected DNA.
To address this concern, we have carried out experiments in which we rescued fusion inhibited by RNAi suppression of La expression by application of recombinant La* (Fig.  S4e). We have now added a representative Western comparing untransfect lysates to lysates from cells transfected with La D371A,D374A, La 1-375, and La* 1-375 that demonstrates the expression of these constructs and the difference in molecular weight between D371A,D374A vs La 1-375. Figure 3 requires further validation. Furthermore, the use of abs to inhibit fusion has to be controlled better. It is not clear that the observed effect is not due to high (background) binding by anti-La, where control abs do not bind, or bind to a much lesser extent.

5) As mentioned above the cell surface staining with anti-La presented in
We appreciate the critique. We validated monoclonal -La mAb by showing lowered cell surface labeling detected with this antibody for the cells with expression of La lowered by siRNA (Fig. S4d). Fig. 3f, -La mAbs inhibited osteoclast fusion and anti-RANK mAbs antibodies had no effect despite comparable levels of binding between -La mAb and -RANK mAb to the surface of synchronized cells following LPC removal (Fig. S5a). Furthermore, -La rabbit Abs ( -La rAb), in contrast to isotype control IgG, also block synchronized osteoclast fusion (Fig. S5b). a-p366 La Ab did not suppress fusion, suggesting that La phosphorylated at Ser366 does not contribute to the fusion stage of osteoclast formation (Fig. S5). Importantly, thanks to the strong, non-specific binding of any rabbit IgG to the abundant Fc receptors on the surface of human macrophage-lineage cells (Ober et al., Int Immunol 13, 1551 (2001), the levels of osteoclast surface binding by all 3 rabbit antibodies: -La rAb, a-p366 La rAb and control IgG, are similar (Fig. S5c). Thus, our finding that only -La rAb inhibits fusion cannot be explained by non-specific steric hindrance of cell surface by associated immunoglobulins.

In the revised manuscript, we present new data arguing against hypothesis that anti-La Abs inhibit fusion because of their high (background) binding. As seen in
6) It remains unclear if the fusion stimulating effect of recombinant La (Fig.4) is exerted on the cell surface and not inside the cell. Certainly, the prolonged (overnight) incubation time with recombinant protein would allow for cellular uptake and a cytosolic effect. Thus, further control experiments are warranted.
In synchronized fusion assay, fusion-stimulating effect of recombinant La and fusioninhibiting effect of anti-La antibody are scored 90 min after application of the protein (Fig.  4e). Note that our conclusion that osteoclast fusion is regulated by cell surface La rather than by intracellular La is supported by several lines of evidence: (i) recombinant La promotes synchronized fusion, Fig. 4e; (ii) anti-La antibodies inhibit synchronized fusion, Fig. 3f; (iii) development of fusion activity of differentiating osteoclasts correlates with an increase in cell surface La staining, Fig. 3d. We consider all these findings and our finding that recombinant full-length La and La 1-375 and La 188-375 promote fusion, La 1-187 has no effect, Fig. 4d difficult to explain within hypothesis that La-derived recombinant proteins and La-targeting antibodies influence fusion by acting inside the cells.

7)
The authors have addressed whether La acts itself as a fusogen. This is described only in the text but the underlying data is not shown/has been omitted?
In the Methods we have more fully the described the assay used: fibroblasts with bound, lipid probe-labeled-erythrocytes, where fusion and even early hemifusion intermediates are detected by the transfer from erythrocyte to fibroblast. In the Results, we more fully describe the data showing that none of 872 analyzed HA0-expressing fibroblasts with bound erythrocyte acquired lipid probe after application of recombinant La in 2 independent experiments. These data indicate that La does not fuse or even hemifuse cells tightly bound by fusion-incompetent hemagglutinin. In the Results, we also present the results of the statistical analysis of this data by S\_fbatf`Xg[bW, suggesting that the probability of Lamediated fusion does not exceed 0.0044 per cell contact. To address this comment of the Reviewer, we added a supplemental figure showing a representative image showing the lack of labeled fibroblasts after application of recombinant La (Fig. S7a). As a positive control, we also included an image showing the very efficient fusion (many labeled fibroblasts) mediated by fusion-competent (=trypsin-cleaved and low pH treated) influenza hemagglutinin.
We have now added new evidence challenging the hypothesis that La acts as an activated fusogen. Application of recombinant La promotes neither lipid mixing of liposomes nor fusion of myoblasts (Fig. S7b,d).

8)
In order to evaluate a role for La as a fusion regulator the authors describe its interaction with Annexin A5 (a protein previously reported by the same lab to be involved in fusion). However, data presented in Fig S5 are not convincing as lysate controls are missing or not run properly alongside the immuno-precipitated samples. The data representing liposome binding experiments (FigS5 D) are confusing in terms of what has been tested but essential negative controls seem to be missing. Also, the primary data (protein gels should be shown).
More importantly however, the authors fail to provide any evidence that would help to answer the question of how the postulated interaction with Annexin A5 would stimulate osteoclast fusion.
As suggested in this comment and discussed in our response to the 2 nd comment 2 of the Reviewer 1, we have deepened our analysis of the La-Anx A5 interactions. Novel and unexpected finding that La binds to Anx A5 at the surface of fusion committed osteoclasts had been supported by following lines of evidence: (1) La-containing protein complexes isolated by immunoprecipitation from osteoclast lysates contain Anx A5; (2) Anx A5containing protein complexes contain La; (3) La binding to liposomes depends on Anx A5, Ca 2+ and PS. Our new findings further substantiate and develop this conclusion: (4) streptavidin pulldown of recombinant 6xHis-La from mixtures of recombinant Biotin-AnxA5 and 6xHis-La; (5, 6) loss of cell surface La observed after lowering the cell surface Anx A5 by either Ca2+ removal or suppressing PS exposure. Our new data provide additional evidence for direct La-Anx A5 binding, explain how La, a soluble protein, associates with cell membrane, and, most importantly, connect La function in osteoclastogenesis with non-apoptotic phosphatidylserine exposure signaling pathways that regulate osteoclast fusion. We consider these findings to be an important step in unraveling the mechanisms by which signaling status of the differentiating osteoclast precursors control the timing and the size of osteoclasts. We have re-written the description of the experiments in which we explore La binding to liposomes via Anx A5 to better explain them.
In response to g[X NXi\XjXetf fcXV\Y\V Vb``Xagf VbaVXea\aZ our immunoprecipitation blots. All blots shown for a particular experiment were ran side by side on the same gels/blots. In these experiments we ran a variety of input samples (e.g., with or without crosslinking) and separated them from our immunoprecipitation lanes by an additional molecular weight standard. Dashed lines simply indicate that we selected lanes of interest and placed them side-by-side.
In general, Anx A5 is an abundant protein in these cells at fusion timepoints. We expect that the majority of Anx A5 is within the cytosol of OCs executing functions that have nothing to do with La. In the immunoprecipitation experiments in Figure S6a, we omitted the input lane so as not to show some large uninterpretable black spot. The point of the Figure S6a is to illustrate that when La or Anx A5 are immunoprecipitated they bring along their binding partner (which does not happen when using isotype control antibodies). We show the input lanes from Anx A1 and Anx A4 simply to demonstrate that our antibodies raised to these proteins work, but that we do not detect these proteins when immunoprecipitating La. At this time, we are unable to comment on the fraction of La that binds Anx A5 or vice versa. We simply demonstrate that these two proteins do interact in this specific biological context and go on to demonstrate that the proteins can directly bind one another in subsequent figures.
Ea eXfcbafX gb g[X NXi\XjXetf specific comment on our liposome binding assays, first we apologize for not explaining the experiments clearly enough to be easily interpreted. Please see where we have amended our previous description of these experiments in our methods section. In addition, please see where we have added a representative gel showing the levels of La or Anx A5 in both soluble and pelleted fractions for the conditions described (Fig.  S6b). 9) Given insufficiently controlled RNAi and anti-La surface binding/ inhibition of fusion experiments, effects observed by the authors on bone resorption (Fig 5) in a osteoblast/osteoclast co-culture setting (Fig 6) and in a mouse model exhibiting an excess of osteoclast fusion (Fig 7), do not really add to the story, as unfortunately they cannot be interpreted with sufficient confidence.

SX TcceXV\TgX g[X NXi\XjXetf VbaVXeaf Uhg [bcX g[Tg TWW\g\baT_
XkcXe\`Xagf iT_\WTg\aZ TaW explaining our approaches have addressed these concerns. As discussed above in our response to the 2 nd comment of this Reviewer, we consider the anti-La antibody used in Fig.  5, 6 and 7 ( -La mAb, Abcam, 75927) to be well validated, including by the new experiments confirming the suppression of the surface La staining by RNAi and by the fusion rescue experiments validating our RNAi approach (Fig. S4e). Furthermore, both new data and, hopefully, better explanations confirm the specificity of the fusion inhibition by anti-La antibodies. Findings in Fig. 6, 7 and 8 support our conclusion that cell surface La regulates not only cell fusion but also bone resorption, and not only osteoclast formation induced by recombinant RANKL but also osteoclast formation triggered by factors released by osteoblasts both in osteoclast/osteoblast cocultures and in an ex vivo model of fibrous dysplasia.

Reviewer #1 (Remarks to the Author):
In the revised manuscript, the authors performed additional experiments and provided more detailed mechanisms underlying how La and Anx5 may regulate osteoclast fusion.
The authors examined the effect of RNAi suppression of La on the expression levels of other fusogenic factors, and the role of La in myocyte fusion was also addressed in revision.
However, the main message of this manuscript remains unproven. Although the reviewer asked to show the physiological relevance of La in osteoclast fusion in vivo, the authors did not address this most important point.
The authors claimed that it is difficult to examine the role of La in vivo because La also functions as an RNA chaperone, but I do not think it reasonable explanation for not performing the in vivo experiments. It will be possible to generate conditional knockout mice or the mice lacking only fusion-related domain of La, which will make the paper much more convincing.
Given that the main message of this work is that La is important for osteoclast fusion but there is no sufficient evidence proving it, the revised manuscript is not suitable for publication in Nature Communications.

Summary
The authors report that the spatial-temporal regulation of La protein contributes to monocyte-toosteoclast differentiation. While La protein is typically thought to play roles in RNA metabolism, the manuscript describes a new direct or indirect contribution of La protein in osteoclast formation. Upon phosphorylation at S366 and caspase cleavage, a low molecular weight species of La is present at the cell surface. Proteomics identified that the levels of this low-molecular weight species of La correlates with multinucleated osteoclasts. This function appears to be independent of the N-terminal binding domains of La proteins. The authors demonstrate that fusion correlates with the levels of La. Moreover, the authors show an ex vivo model of osteoclast formation in +-'3164 ) 842. &4-& 7-5, /65&5-104 -0 $94# %-5, 5,-4 /1)*." 5,* /&064(3-25 )-42.&84 5,&5 5,* application of anti-La antibodies can reduce osteoclast fusion, suggesting a possible use of La protein as a potential therapeutic target. While some of the findings in the paper are mostly correlative and must be presented carefully as such, the possible implications are of interest.
The authors have addressed my major concerns regarding the manuscript.
Reviewer #5 (Remarks to the Author): Comments: The authors present very interesting observations suggesting a new functional role for La in osteoblast fusion by using various experimental approaches. The findings are relevant and of interest to the field. Although the quality of the manuscript probably improved after revision, some experiments and data are still lacking clarity and convincing conclusions.
The identification of La as the protein species within the gel band is great, but the information provided within the manuscript is not sufficient. Shot gun proteomic analyses of gel bands from complex lysates usually result in the identification of many proteins, and certain filter, such as protein abundance rank, peptide number (unique peptides!), coverage, enrichment over control band etc, have to be applied for solid matching and target identification. Were La and Vimentin the only proteins identified? Was La the most abundant protein? Based on the staining signal, La should be much more abundant compared to Vimentin. Is this reflected in the mass spec data?
From the current version of the manuscript it is not clear, how the mass spec data (protein lists) were analyzed/filtered after RAW data analyses. More details would be very much appreciated.
The binding of La to AnxA5 is a major result, but despite the effort to show evidences for direct b,d), data still lack strength. The AnxA5 Co-IP experiment with La ( Figure 5a) would benefit from additional controls. Do the functionally different or similar La variants (1-187,  bind Anx5A? Does Anx5A binding correlate with function in osteoblast fusion? The western blot signal of La Co-IP with biotinylated AnxA5 in Fig 5b is not very convincing. A proper control (unrelated biotinylated protein, for example Anx1 or Anx4) should be included in order to show specificity. The La western blot signal in Figure S6a (Anx5A pulldown) is not very convincing, neither. Data presentation should be improved. Is the signal with IgG control background or La specific? The liposome assay (Figure 5c, S6b) seems to be an elegant assay, but lacks controls (for example nonfunctional La 1-188 construct, other annexins) in order to be completely convincing. The Anx5 depletion essay (Fig 5g) as an approach to confirm La interaction and binding on the cell surface is rather indirect and lacks experimental design. EGTA and A01 treatment (Fig5e,f) leads to decrease in AnxA5 and La cell surface signal. The AnxA5 dependency of La surface binding is not shown. La itself could be Calcium and PS dependent. The experiment should be expanded, adding recombinant La protein, even including non-functional La constructs (1-188), to EGTA/A01 treated (AnxA5 depleted) cells.
The lack of La in mediating myoblast cell fusion ( Fig S7) is a quite relevant and interesting observation, and actually provides tools to further investigate the function of La in cell fusion. How do the authors explain this observation? Is La expressed in myoblasts, but not surface localized? Does recombinant La actually binds to AnxA5 and/or to myoblast cell surface? As far as I understand the figures, this was not shown. These questions can be easily addressed, for example by immunostaining and outcome is actually crucial for interpretation of data presented in this study (results of anti La mAB and recombinant La). Assuming La and Anx A5 are expressed in myoblasts, they seem not to be enough to drive cell fusion and additional factors are required. Here an unbiased proteomic approach, such as affinity pulldown combined with mass spectrometry, would allow characterization of La interactomes of functionally different La variants as well as in different cellular background.
The authors should be careful with the interpretation of a-p366 La antibody results ( Figure S5b,c). This antibody binds to La when phosphorylated at position 366. According to statements within this study phosphorylated La exists as full length protein and exclusively localizes to the nucleus, therefore it is not cell surface exposed. Although the authors state on page 10 lane 2, that antibody binding was similar, including anti p366 La antibody, the data for that phosphorylation specific antibody are not shown (surface staining), and to my understanding phosphorylated La is not supposed to localize at the membrane. First, I wonder what the rational was for using the anti p-366 La antibody in this essay? Using the anti La mAB as an additional antibody would have made more sense, since at least it recognizes the surface exposed La variant. Second, interpretations should be done carefully. Making conclusion about phosphorylation status is not appropriate and rather indirect, since phosphorylated La was not shown to be surface localized so far, and impact of phosphorylation (for example using cleavage mutant with phosphomimetic mutation) was not investigated.
Specific comments: Images and western blots for La in murine cells (Figure 2a/b) show no nuclear localization of La as well as only one La species at day 3, in contrast to human cells, where an higher molecular weight La species is accumulated in the nucleus at later time points. How do the authors explain that difference?
In Figure S3a, western blot signal of La in untreated cells shows strong full length La signal, but the corresponding images do not show any nuclear signal, which is inconsistent with data from Figure 1g, where at day 3 nuclear signal of La was observed. How can this be explained?
Minor: When addressing La phosphorylation, the authors should call it non-phosphorylated La, since dephosphorylation implies a phosphorylation event before that, which might not be the case here. It rather seems to be a La post-translational modification happening in the nucleus.
Reviewer #6 (Remarks to the Author): In the revised manuscript, the authors have addressed most of the questions by Reviewer #4. There are only a few minor points that need to be clarified: 1. The size of La in human osteoclasts . -In Figure 1c, the band used for mass spec was above 50 kDa and the identified protein was La. However, in Figures 1e, both FL and LMW La proteins were below 50 kDa . -In Figure 1d, two bands were in the monocyte lysate lane. Were they FL and LMW La proteins, or the FL La and a non-specific upper band? If the former, did the author detect significant cytoplasmic staining of La in the monocyte? If the latter, could the authors provide some explanation on the absence of the non-specific band in the other two lanes, as well as the absence of LMW La in the RANKL sample? 2. SSB expression and La protein level in the M-CSF sample . -In Figure S1e, there were ~2 fold SSB transcripts in the M-CSF than the RANKL sample, but there was no detectable La protein in the M-CSF sample (Figure 1d). Is this difference due to translation inhibition or protein degradation in the M-CSF sample? -In Figure 2e, a D1 sample after RANKL treatment should be included. 3. Localization of FL La vs. p366 La . -In Figure S3, there was no LMW La and strong p366 La in the nucleus after caspase inhibitor treatment. Is the FL La also only localized in the nucleus on Day 3? 4. Localization of Anx A5, La and PS . -In Figures 5 and S6, in the absence of Ca2+, no Anx A5 associated with liposome but 15% of La did. Does this mean that some La can interact with PS in the absence of AnxA5? -The immunofluorescent staining showed strong Anx A5 surface staining (Figure 5e) but weak La staining. Do these proteins co-localize? -In Figure 5e, does the strong staining of Anx A5 suggest that the cell surface of human OCs is enriched with PS without inducing apoptosis?